MiR-140 Inhibits The Invasion And Migration Of Lung Adenocarcinoma A549 Cells By Down Regulating Smad3 Expression

Background: mi RNA(micro RNA,micro RNA)is a type of single-stranded non-coding small molecule RNA encoded by an endogenous gene.It consists of 19 to 24 base pairs(bp).It recognizes the target m RNA(messenger RNA)and completely or partially complements the 3’UTR of the target m RNA.Furthermore,it exerts its function of degrading or inhibiting the translation of target m RNA,and regulates the expression of target genes,thereby affecting cell proliferation,differentiation,metabolism,and apoptosis.Recent studies have found that mi RNAs can be used as oncogene or tumor suppressor gene to participate in the biological process of tumor cell proliferation,invasion and metastasis.mi R-140(micro RNA-140)is a mi RNA specifically expressed in cartilage tissue.The decrease in expression is related to the occurrence of osteoarthritis and plays an important role in the process of cartilage tissue proliferation and development.More and more studies show that mi R-140 is involved in the occurrence and development of multiple tumor types,but its underlying mechanism of action is still unclear and controversial.Studies have confirmed that Smad3 protein is the first transcription factor transmitted by the TGF-β signaling pathway,can significantly promote the invasion and metastasis ability of breast cancer,ovarian cancer,non-small cell lung cancer,current research has confirmed that Smad3 protein is a direct target of mi R-140.Therefore,mi R-140 may inhibit the migration and invasion of lung adenocarcinoma A549 cells by down regulating the Smad3 protein.Objective: This experiment first validated Smad3 protein as the target of mi R-140,and further explored the effect of mi R-140 on the invasion and migration of lung adenocarcinoma A549 cells,and clarifies the relationship between Smad3 protein and mi R-140,in order to providing new candidate targets for the diagnosis and treatment of lung cancer.Methods:1.Up regulation group: transfection of mi R-140 mimics,mi R-140 unrelated sequences(mimics-NC),and Smad3 small interfering RNA(si RNA-Smad3)into human adenocarcinoma of lung A549 cellsvia lipofectamine 2000;Down regulation group: transfection of mi R-140 inhibitor and mi R-140 unrelated sequences(inhibitor-NC)into A549 cells via liposomes.2.Real-time fluorescence quantitative(q RT-PCR)was used to detect the expression levels of mi R-140 and Smad3 m RNA in A549 cells.Western blot was used to detect the expression level of Smad3 protein.The relationship between mi R-140 and Smad3 protein was analyzed.3.Apply scratch test,transwell chamber to detect the effect of mi R-140 up regulation and down regulation on A549 cell invasion and migration ability,and the effect of Smad3 down regulation on A549 cell invasion and migration ability,to analyze the regulatory mechanism of mi R-140 on lung adenocarcinoma A549 cells.4.SPSS19.0 software was used for statistical analysis.P <0.05 was considered statistically significant.Results:1.The results of q RT-PCR experiments showed that mi R-140 was significantly expressed in A549 cells transfected with mi R-140 mimics compared with the control group(P <0.05),indicating successful transfection.Western blot results showed that the expression of Smad3 protein in mi R-140 mimics group was significantly lower than that in the control group(P <0.05).2.The results of cell scratch test showed that the migration ability of A549 cells in mi R-140 mimics group was significantly reduced compared with the control group(P <0.05),but there was no significant difference compared with the si RNA-Smad3 group(P>0.05).Transwell chamber invasion experiment results showed that the number of transmembrane cells in the mi R-140 group was significantly reduced compared to the control group(P<0.05),but not significantly different from the si RNA-Smad3 group(P>0.05).3.In contrast,the expression level of Smad3 protein in the mir-140 inhibitor group was significantly higher than that in the control group(P< 0.05).Cell scratch experiments showed that the migration ability of A549 cells in the mir-140 inhibitor group was significantly higher than that of the control group(P<0.05),while the migration ability of the sirna-smad3 + mir-140 inhibitor group was not significantly changed compared with that of the control group(P>0.05).Transwell invasion showed that the number of membrane-penetrating cells of the mir-140 inhibitor group was significantly increased compared with the control group(P < 0.05),while the migration capacity of the sirna-smad3 + mir-140 inhibitor group was not significantly changed compared with the control group(P>0.05).Conclusion:1.Up regulating mi R-140 can reduce the expression of Smad3 protein,On the contrary,down-regulating mi R-140 can increase the expression of Smad3 protein,which confirms that Smad3 protein is indeed the target of mi R-140.2.Up regulation of mir-140 and inhibition of Smad3 expression alone could inhibit the invasion and migration of A549 cells.Down regulation of mir-140 can promote the expression of Smad3 protein and promote the invasion and migration of A549 cells.Down regulation of mir-140 and inhibition of Smad3 expression had no significant effect on the invasion and migration of A549 cells.Therefore,mir-140 can down regulate the expression of Smad3 and inhibit the invasion and migration of lung adenocarcinoma A549 cells.3.Low expression of mi R-140 and high expression of Smad3 are related to invasion and migration of lung adenocarcinoma A549 cells.mi R-140 may become a candidate target for lung cancer treatment,providing new ideas for the treatment of lung cancer.